PRO PROJECT SUMMARY Macrophages (MO) play a crucial role in the propagation of inflammation in many diseases. Recent work has highlighted functional MO heterogeneity in synovial tissue of rheumatic diseases. The presence of activated MO and the imbalance of pro-inflammatory and anti-inflammatory MO populations in the synovium has been related to disease progression and clinical manifestations. Among other stimuli, MO in the joints are activated by damage associated molecular patterns (DAMP), that are sensed by NLRP3 inflammasome and activate IL-1? and IL-18- dependent inflammation that shifts joint cells from a resting regulatory state to a highly metabolically active one. Abnormal choline metabolism is emerging as a metabolic hallmark of cell activation and inflammation. Choline is a vitamin-like essential nutrient that is phosphorylated by choline kinase alpha (ChoK) as precursor to newly synthesized phosphatidylcholine (PC). Emerging lipidomic studies indicate that some rheumatic diseases exhibit an increase of choline circulating levels and an altered phospholipid (PL) profile in the synovial fluid. In addition, synovial MO express choline transporters, suggesting a role of choline metabolism in synovial MO activation. Previous work by the PI has established an essential role for the vitamin-like nutrient choline uptake and mobilization towards PC synthesis and NLRP3 inflammasome-dependent production of IL-1? and IL-18 in activated MO. Inhibition of choline uptake or phosphorylation by ChoK altered mitochondrial PL content and reduced cellular ATP, which resulted in the initiation of mitophagy, prevention of mitochondrial DNA oxidation, and ultimately, prevention of activation of NLRP3 inflammasome and production of IL-1? and IL-18. In addition, ChoK inhibition decreased inflammation in murine models of gout and Muckle Wells syndrome. The PI now provides preliminary data suggesting that choline utilization in MO modulates MO biology including MO polarization. She also describes that LPS-induced MO activation is accompanied by metabolic and PL reorganization changes that are dependent on choline availability. Additionally, she shows novel data about the role of ChoK in osteoarthritis. These findings suggest the attractive hypothesis that nutrient metabolism could modulate synovial activated MO phenotypes in the context of rheumatic diseases. The current proposal will provide a comprehensive vision of the role of choline metabolism and PL composition in MO activation and biology, through three Specific Aims. Aim 1 will evaluate choline metabolism in MO differentiation and polarization; Aim 2 will explore the role of choline availability on their associated metabolic and bioenergetic changes; Aim 3 will investigate choline availability and its phosphorylation in the OA synovium. The proposed studies and training plan will help PI?s transition to an independent investigator and will provide her with expertise in immunometabolism, translational research, metabolomics, and mitochondrial bioenergetics. The work will be performed with the mentorship of Dr. Michael Karin, an expert in myeloid biology, and Dr. Monica Guma, an expert in arthritis research, as well as guidance from a stellar Advisory Committee.